The therapeutic potential of miRNAs in cardiac fibrosis: where do we stand?

Recent developments in basic and clinical science have turned the spotlight to miRNAs for their potential therapeutic efficacy. Since their discovery in 1993, it has become clear that miRNAs act as posttranscriptional regulators of protein expression. Their clinical potential was further highlighted by the results of miRNA-based interventions in small laboratory animals. More importantly, their therapeutic effectiveness has been shown recently in phase 2a clinical studies in patients with hepatitis C virus infection, where inhibition of miRNA-122 showed prolonged and dose-dependent viral suppression. A recent study surprisingly revealed the presence of plant-derived miRNAs in the blood of healthy humans. This finding opens up the possibility to explore miRNA-mediated therapeutics derived from (genetically modified) food. Having arrived at this point in our understanding of miRNAs, we provide an overview of current evidence and future potential of miRNA-based therapeutics, focusing on their application in cardiac fibrosi

Repression of Cardiac Hypertrophy by KLF15: Underlying Mechanisms and Therapeutic Implications

The Kruppel-like factor (KLF) family of transcription factors regulates diverse cell biological processes including proliferation, differentiation, survival and growth. Previous studies have shown that KLF15 inhibits cardiac hypertrophy by repressing the activity of pivotal cardiac transcription factors such as GATA4, MEF2 and myocardin. We set out this study to characterize the interaction of KLF15 with putative other transcription factors. We first show that KLF15 interacts with myocardin-related transcription factors (MRTFs) and strongly represses the transcriptional activity of MRTF-A and MRTF-B. Second, we identified a region within the C-terminal zinc fingers of KLF15 that contains the nuclear localization signal. Third, we investigated whether overexpression of KLF15 in the heart would have therapeutic potential. Using recombinant adenoassociated viruses (rAAV) we have overexpressed KLF15 specifically in the mouse heart and provide the first evidence that elevation of cardiac KLF15 levels prevents the development of cardiac hypertrophy in a model of Angiotensin II induce

Differential regulation of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments

Steinbusch LK, Wijnen W, Schwenk RW, Coumans WA, Hoebers NT, Ouwens DM, Diamant M, Bonen A, Glatz JF, Luiken JJ. Differential regulation of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments. Am J Physiol Cell Physiol 298: C1549-C1559, 2010. First published April 7, 2010; doi:10.1152/ajpcell.00334.2009.-Insulin and contraction stimulate both cardiac glucose and long-chain fatty acid (LCFA) uptake via translocation of the substrate transporters GLUT4 and CD36, respectively, from intracellular compartments to the sarcolemma. Little is known about the role of vesicular trafficking elements in insulin-and contraction-stimulated glucose and LCFA uptake in the heart, especially whether certain trafficking elements are specifically involved in GLUT4 versus CD36 translocation. Therefore, we studied the role of coat proteins, actin-and microtubule-filaments and endosomal pH on glucose and LCFA uptake into primary cardiomyocytes under basal conditions and during stimulation with insulin or oligomycin (contractionlike AMP-activated protein kinase activator). Inhibition of coat protein targeting to Golgi/endosomes decreased insulin/oligomycinstimulated glucose (- 42%/-51%) and LCFA (-39%/-68%) uptake. Actin disruption decreased insulin/oligomycin-stimulated glucose uptake (-41%/-75%), while not affecting LCFA uptake. Microtubule disruption did not affect substrate uptake under any condition. Endosomal alkalinization increased basal sarcolemmal CD36 (2-fold), but not GLUT4, content, and concomitantly decreased basal intracellular membrane GLUT4 and CD36 content (-60% and -62%, respectively), indicating successful CD36 translocation and incomplete GLUT4 translocation. Additionally, endosomal alkalinization elevated basal LCFA uptake (1.4-fold) in a nonadditive manner to insulin/oligomycin, and decreased insulin/oligomycin-stimulated glucose uptake (-32%/-68%). In conclusion, 1) CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process depending on coat proteins, and 2) GLUT4 and CD36 trafficking are differentially dependent on endosomal pH and actin filaments. The latter conclusion suggests novel strategies to alter cardiac substrate preference as part of metabolic modulation therapy

Attenuation of cardiac hypertrophy in AAV9-KLF15 mice.

<p><b>a</b>) Body weight is not different in sham and AngII treated animals. <b>b</b>) AngII induces hypertrophy in GFP expressing mice, as measured by correcting the left ventricular weight (LVw) for body weight (Bw). This effect is blunted in mice with cardiac specific overexpression of KLF15. <b>c and d</b>) The hypertrophic response was also measured by analysis of individual cardiomyocyte size. Mice overexpressing KLF15 showed a blunted hypertrophic response to AngII. AU: arbitrary units. Scale bar in d represents 50 µm <b>e</b>) Quantitative real-time PCR on left ventricular tissue showed an increased expression of the hypertrophic markers ANF and alpha skeletal actin (aSKA) in GFP mice treated with angiotensin. In mice that overexpress KLF15 we found a reduction in expression of these genes, but this is not significant. * p<0.05 compared to GFP control. # p<0.05 compared to GFP ANGII.</p

KLF15 represses and binds MYOCD and MRTF-A and –B.

<p>(<b>a</b>) schematic overview of MYOCD, MRTF-A and MRTF-B. All the proteins share a SRF binding and a (potential) KLF15 binding domain. (<b>b</b>) KLF15 represses MRTF-A and MRTF-B mediated activation of the SRF responsive −505 Sm22 luciferase reporter. (<b>c</b>) KLF15 represses MRTF-A and MRTF-B mediated activation of the SRF responsive −638 ANF luciferase reporter. (<b>d</b>) A GST-pulldown assay using in vitro translated ³⁵S labeled MRTF-A and MRTF-B and GST-fused KLF15 shows a direct interaction between KLF15 and MRTF-A and –B. (<b>e</b>) GST pulldown assays using ³⁵S labeled SRF and GST fused KLF15 shows no interaction between KLF15 and SRF. (<b>f</b>) KLF binding site (CACCC) in the −505 Sm22 reporter. This bindingsite is mutated to a TGTTT site. (<b>g</b>) deletion of the KLF binding site in the −505 Sm22 reporter does not affect the repressive effect of KLF15.</p

Cellular distribution of full-length and truncated KLF15 proteins.

<p>To determine which part of the KLF15 protein contains the nuclear localization signal (NLS) several truncated KLF15 open reading frames were cloned into an expression vector containing an N-terminal FLAG tag and transfected into COS-7 cells. (<b>a</b>) A schematic overview of the KLF15 truncated proteins. Full length murine KLF15 has a length of 416 aa and contains three highly conserved zinc fingers (Zn1, Zn2 and Zn3) that are located at the C-terminal end of the protein (aa315-aa416). (<b>b</b>) COS-7 cells transfected with the KLF15 expression plasmids were fixed and immunostainings were performed using a primary antibody against the FLAG-tag and a fluorescent secondary antibody (Alexa 488). Nuclei were stained with DAPI. Full length KLF15 (1–416 aa) is localized in the nucleus. Truncated KLF15 proteins lacking the NLS (1–205 aa and 1–315 aa) fail to translocate to the nucleus. All KLF15 mutant proteins lacking N-terminal parts, but containing the three zinc fingers (102–416 aa, 199–416 aa, 308–416 aa) are located in the nucleus. A KLF15 mutant lacking Zn2 and Zn3 is not located in the nucleus indicating that both Zn2 and Zn3 are necessary for nuclear localization of KLF15. (<b>c</b>) To study whether the three zinc fingers of KLF15 can acts as a NLS we fused one or more zinc fingers to eGFP. A schematic representation of the four eGFP constructs that were used are shown. (<b>d</b>) eGFP is both localized in the cytosol and the nucleus. When all three zinc fingers are fused to eGFP, eGFP is restricted to the nucleus, indicating that the three zinc fingers are sufficient to drive nuclear localization. When eGFP is only fused to Zn2 and Zn3, expression is still nuclear, but when fused to Zn3, expression is both cytosolic and nuclear, indicating that Zn2 and Zn3 act as NLS.</p

In vivo overexpression of FLAG-KLF15 using AAV9.

<p><b>a</b>) Western blot analysis of left ventricular samples revealed expression of FLAG-KLF15 protein. <b>b</b>) Experimental timeline. Eight week old mice are intravenously injected with 1*10¹⁰ vector genomic copies AAV-9 (KLF or GFP). One week later hypertrophy is induced by implanting osmotic minipumps that release 1.5 µg/g/day of angiotensin II. Four weeks later, mice were sacrificed.</p

Regulation of Cardiac Gene Expression by KLF15, a Repressor of Myocardin Activity*

Pathological forms of left ventricular hypertrophy (LVH) often progress to heart failure. Specific transcription factors have been identified that activate the gene program to induce pathological forms of LVH. It is likely that apart from activating transcriptional inducers of LVH, constitutive transcriptional repressors need to be removed during the development of cardiac hypertrophy. Here, we report that the constitutive presence of Krüppel-like factor 15 (KLF15) is lost in pathological hypertrophy and that this loss precedes progression toward heart failure. We show that transforming growth factor-β-mediated activation of p38 MAPK is necessary and sufficient to decrease KLF15 expression. We further show that KLF15 robustly inhibits myocardin, a potent transcriptional activator. Loss of KLF15 during pathological LVH relieves the inhibitory effects on myocardin and stimulates the expression of serum response factor target genes, such as atrial natriuretic factor. This uncovers a novel mechanism where activated p38 MAPK decreases KLF15, an important constitutive transcriptional repressor whose removal seems a vital step to allow the induction of pathological LVH

MiR30-GALNT1/2 Axis-Mediated Glycosylation Contributes to the Increased Secretion of Inactive Human Prohormone for Brain Natriuretic Peptide (proBNP) From Failing Hearts

Recent studies have shown that plasma levels of the biologically inactive prohormone for brain natriuretic peptide (proBNP) are increased in patients with heart failure. This can contribute to a reduction in the effectiveness of circulating BNP and exacerbate heart failure progression. The precise mechanisms governing the increase in proBNP remain unclear, however. We used our recently developed, highly sensitive human proBNP assay system to investigate the mechanisms underlying the increase in plasma proBNP levels. We divided 53 consecutive patients hospitalized with heart failure into 2 groups based on their aortic plasma levels of immunoreactive BNP. Patients with higher levels exhibited more severe heart failure, a higher proportion of proBNP among the immunoreactive BNP forms secreted from failing hearts, and a weaker effect of BNP as estimated from the ratio of plasma cyclic guanosine monophosphate levels to log-transformed plasma BNP levels. Glycosylation at threonines 48 and 71 of human proBNP contributed to the increased secretion of proBNP by attenuating its processing, and GalNAc-transferase (GALNT) 1 and 2 mediated the glycosylation-regulated increase in cardiac human proBNP secretion. Cardiac GALNT1 and 2 expression was suppressed by microRNA (miR)-30, which is abundantly expressed in the myocardium of healthy hearts, but is suppressed in failing hearts. We have elucidated a novel miR-30-GALNT1/2 axis whose dysregulation increases the proportion of inactive proBNP secreted by the heart and impairs the compensatory actions of BNP during the progression of heart failur